Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the ²⁶² RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene ( NEO ) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro . After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500–1,250. These nonintegrative vectors were also efficient in vivo , allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.

Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.

Objective To describe clinical and radiologic features of cranial nerve (CN) involvement in patients with myelin oligodendrocyte glycoprotein antibodies (MOG-IgG) and to assess the potential underlying mechanism of CN involvement using a nonhuman primate (NHP) model. Methods Epidemiologic, clinical, and radiologic features from a national cohort of 273 MOG-IgG–positive patients were retrospectively reviewed for CN involvement between January 2014 and January 2018. MOG-IgG binding was evaluated in CNS, CN, and peripheral nerve tissues from NHP. Results We identified 3 MOG-IgG–positive patients with radiologic and/or clinical CN involvement. Two patients displayed either trigeminal or vestibulocochlear nerve lesions at the root level, and the remaining patient had an oculomotor nerve involvement at the root exit and at the cisternal level. Additional CNS involvement was found in all 3 patients. None of the 3 patients9 sera recognized MOG expression in CN of NHP. Conclusion Craneal nerve involvement can coexist in patients with MOG antibody disease, although the underlying pathophysiology remains elusive.

Lentiviral vectors are among the most efficient gene transfer tools for dividing and non-dividing cells. However, insertional mutagenesis has been observed in clinical trials with oncoretroviral vectors and this has prompted detailed study of genotoxicty of all integrating vectors. For many applications, avoiding integration is the most straightforward approach to overcome this problem and is facilitated by the extensive studies of the integrating mechanisms of lentiviruses. Indeed, non-integrating lentiviral vectors have been developed by mutating the integrase gene or by modifying the attachment sequences of the LTRs. In this review, we first consider on the toxicity associated with integration and on lentivirus integrase biology, and discuss the implications of integrase mutant studies for the development of non-integrating lentiviral vectors. We review published data concerning non-integrating lentiviral vectors with particular focus on their residual integration and transgene expression efficiency. Finally, the latest advances in the development of genetic engineering tools derived from non-integrating lentiviral vectors are presented.